Project Summary/Abstract G protein-coupled receptors (GPCRs) assemble as oligomers that determine their distinct functional properties. However, we actually know little about how these important receptors assemble, or how lipids in the plasma membrane, where most GPCRs function, modulate their structure and function. What determines the selectivity of GPCRs towards lipids, and the coupling between oligomerization and lipid/ligand binding events remains a key knowledge gap in the field; one that if addressed will significantly advance our understanding of how these receptors participate in both normal and pathophysiological processes. For more than three decades, an arsenal of biochemical approaches has been applied to investigate GPCR structure, indicative of the importance of this superfamily of receptors. However, because biochemical data largely constitutes indirect evidence on structure, there exists a controversy in the field as to whether GPCRs function as dimers or larger, and more complex, oligomers. Here, we propose to address this long-standing debate by adapting new ion mobility mass spectrometry (IM-MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer, to determine the stiochiometry and distribution of GPCR oligomers. More specifically, we will apply novel IM-MS technology we have pioneered to resolve and interrogate individual lipid and ligand binding events to membrane protein complexes. Unlike other biophysical approaches, these methods allow us to determine the effects of ligands on GPCR monomers and oligomers with unparalleled resolution. As a model GPCR, we propose to work on the rat neurotensin receptor type 1 (NTSR1), and have obtained proof-of- principle data using this receptor bound to agonist (neurotensin peptide). The native mass spectrum reveals two populations of oligomeric complexes, with one of them matching the anticipated one to one complex. Interestingly, the second complex revealed a unique stoichiometry, departing from previous reports of NTSR1 as being dimeric. Our long-term goal is to test the hypothesis that GPCR oligomers are dynamic and modulated by bound ligand and lipid. It is the objective of this R21 application to lay the foundation for those studies by optimizing purification of NTSR1 specifically for IM-MS to allow us for the first time to resolve individual ligand and lipid binding events to NTSR1 monomers and oligomers. We then propose to determine the influence of different detergent environments on the oligomeric states of apo and agonist-bound NTSR1 to lay the groundwork for future studies of how bound ligands determine how NTSR1 and other GPCRs assemble and function.